Antioxidant Potential and Polyphenol Content of Beverages, Chocolates, Nuts, and Seeds

Abstract

Polyphenols present in plant foods as natural antioxidants may inhibit the pathogenesis of many diseases, which involve oxidative reactions. This study aimed to measure the total antioxidant potential with the ferric reducing antioxidant power method and the total polyphenol contents of 15 kinds of beverages, 4 types of chocolates, and 6 sorts of nuts and seeds commonly consumed. The antioxidant potential and polyphenol content ranged (respectively): in beverages 0.216–2.940 mmol/100 ml and 31–241 mg/100 ml, in chocolates 0.550–14.67 mmol/100 g and 222–1617 mg/100 g, in nuts and seeds 0.851–55.91 mmol/100 g and 125–3521 mg/100 g. The antioxidant potential of the foods tested was related to the total polyphenol contents.

Keywords:

• Antioxidant potential
• Polyphenols
• FRAP
• Beverages
• Chocolate
• Nuts
• Seeds

INTRODUCTION

Numerous epidemiologic studies suggest that regular consumption of foods and beverages abundant in polyphenols is associated with a decreased risk of deleterious processes in the body induced by the oxidative stress.[1–3 −3 Polyphenols are the largest group of natural antioxidants in the diet. The average daily intake of polyphenols is estimated to be approximately 1 g, which is more than the intake of known dietary antioxidants, such as vitamins and minerals, taken altogether.[4]

A regular fluid intake from a daily diet is recommended to maintain good health; therefore, beverages are an inseparable part of the human diet. Beverages are the main dietary sources of polyphenols and the largest contributors to the total dietary antioxidant capacity.[5] The polyphenols from beverages are completely bioaccessible, because they pass directly into the intestinal fluids.[6] Patterns of beverage consumption vary widely throughout the world. For example, primary beverages consumed in Spain are coffee, beer, and wine, while it is coffee in Finland and tea in Poland, the United States, Japan, and China.[7–9 −9 In Europe and the United States, black tea is the most common tea beverage consumed, while green tea is the most common tea in China and Japan.[10]

Compared to beverages, consumption of chocolate, nuts, and seeds is rather sporadic, but these foods abound in many compounds, which demonstrate beneficial effects on human health. Chocolate, nuts, and seeds are rich in polyphenols; therefore, even moderate consumption of these products may significantly influence antioxidant properties of a diet.[11] Some studies indicate that the supplementation of a human diet with nuts increases blood plasma antioxidant potential and manifests beneficial effects on lipid profiles.[12]

We previously determined the total polyphenol content and antioxidant activity in vegetables, pulses, fruits, and cereal products commonly consumed in Poland.[13] This present survey was a follow-up of the previous study with an aim to evaluate total polyphenol contents and antioxidant activities in beverages, chocolates, nuts, and seeds. The results of the present work may be helpful to categorize foods according to antioxidant activity and to supplement the already existing food databases.

MATERIALS AND METHODS

Food Samples

Twenty-five food products (15 kinds of beverages, 4 types of chocolates, and 6 sorts of nuts and seeds) were randomly purchased in triplicate at different local food markets. Edible parts of nuts and seeds (approximately 100 g) were dried using a convection air-oven (BMT, Brno, Czech Republic) at 120°C for 60 min.[14] Dried products were pulverized in a grinder and stored at room temperature in a desiccator in plastic containers until analysis.[15] Tea, coffee, drinking chocolate, hot cocoa, and chocolates were analyzed individually. Fruit juices, wine, and beer were analyzed directly.

Food Extraction

Pulverized samples (0.25 g) of nuts and seeds or 1 g of chocolates dissolved in 5 ml of hot distilled water were placed in test tubes with a mixture of 10 ml of methanol/water (50:50, v/v). The pH was adjusted to 2 using 2M HCl. The tubes were thoroughly shaken at room temperature for 1 h, and then centrifuged at 4000g for 10 min. Supernatants were collected in clean dry test tubes. Then the residues were extracted again with 10 ml of an acetone/water mixture (70/30, v/v). Tea, coffee, and hot cocoa infusions were prepared as follows: 1 g tea, coffee, or hot cocoa was extracted for 3 min with 100 ml of boiling distilled water. Drinking chocolate (1 g) was dissolved in 100 ml of hot distilled water. The methanol-acetone extracts of nuts, seeds, and chocolates and water extracts of beverages were used to determine the total polyphenol content and the antioxidant activity.

Total Polyphenol Contents

Total polyphenol contents of foods were determined with the Singleton-Rossi method.[16] As described previously, 0.2 ml of a sample was mixed with 1 ml Folin-Ciocalteau reagent previously diluted in distilled water (1:10) and 0.8 ml of 7.5 % (w/v) sodium carbonate.[13] The absorbance was measured after 30 min at 765 nm on a Spekol 10 spectrophotometer (Carl Zeiss, Jena, Germany). The results were expressed as gallic acid equivalents (GAE).

Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP was determined according to Benzie and Strain.[17] A 1.5 ml of 2,4,6-tripyridyl-s-triazine (TPTZ) solution was warmed to 37°C, then a reagent blank was measured at 593 nm on a Spekol 10 spectrophotometer (Carl Zeiss, Jena, Germany). Subsequently, 50 μl of the sample dissolved in distilled water (1:5) was added to the FRAP reagent. The absorbance was measured following incubation at 37°C for 4 min. The antioxidant potential of samples was determined from the standard curve using FeSO₄•7H₂O at a concentration range between 100 and 1000 μmol/l. The presented data are the means of two measurements.

Statistical Analysis

Results were expressed as mean values ± standard deviations. Correlations between variables were calculated with the Pearson's test. Results were considered statistically significant at p < 0.05. The data analysis was performed using Statistica 9.0 software (StatSoft, Inc., Poland).

RESULTS AND DISCUSSION

Beverages

The antioxidant activities and total polyphenol contents in the studied beverages are shown in Table 1. The wines were characterized by varied antioxidant activities and polyphenol contents. The highest FRAP antioxidant activity was found in red wines—2.940 ± 0.40 mmol/100 ml, at the polyphenol content 241 ± 32 mg/100 ml. The lowest FRAP activity (0.250 ± 0.11 mmol/100 ml), as well as the polyphenol contents among wines was observed in white wine (26 ± 9 mg/100 ml). These findings are in agreement with other studies.[18] Wines contain a variety of polyphenolic compounds, the most abundant being anthocyanins, which mostly originate from the skin of grape berries. The polyphenols are important for the sensoric properties of wines, especially affecting the taste and color of red wine. Some studies[19] showed that red wines manifested higher FRAP properties than white wines, and that dry wines had higher FRAP than sweet ones. A moderate red wine consumption significantly increases concentration of total polyphenols in the human plasma having beneficial effects on the antioxidant status and indices of lipid peroxidation, which are associated with coronary heart disease.[20]

Table 1  Total polyphenol content and antioxidant activity in beverages in order of the FRAP antioxidant activity

Beverages	N	Polyphenol content (mg/100 ml)	FRAP (mmol/100 ml)
Red wine	3	241 ± 32	2.940 ± 0.40
Blackcurrant juice	3	108 ± 3	1.477 ± 0.07
Instant coffee	3	133 ± 9	1.417 ± 0.12
White tea	3	104 ± 5	1.072 ± 0.19
Green tea	3	85 ± 3	0.890 ± 0.16
Black tea	3	72 ± 15	0.783 ± 0.14
Orange juice	3	72 ± 1	0.673 ± 0.16
Drinking chocolate	3	60 ± 7	0.573 ± 0.07
Ground coffee	3	53 ± 7	0.562 ± 0.08
Rooibos tea	3	54 ± 2	0.452 ± 0.04
Red tea	3	38 ± 3	0.346 ± 0.06
White wine	3	26 ± 9	0.250 ± 0.11
Apple juice	3	40 ± 2	0.243 ± 0.02
Hot cocoa	3	30 ± 7	0.237 ± 0.08
Beer	3	31 ± 1	0.216 ± 0.05
N = number of samples.

Among the beverages tested, the instant coffee displayed good antioxidant properties at considerable polyphenol contents (1.417 ± 0.12 mmol/100 ml and 133 ± 9 mg/100 ml, respectively), which was in concordance with other authors.[21] Coffee is generally processed by many ways, including fermentation, roasting, grounding, and brewing, which influence the final concentration of polyphenols in coffee beverages, particularly the major components—chlorogenic, caffeic, and ferulic acids. In this study, the total polyphenols content in brewed instant coffee was almost 3-fold higher than in the brewed ground coffee, which can be associated with the specific method of instant coffee production. Some authors indicate that phenolic acids present in coffee exhibit scavenging activity against reactive oxygen species.[22,23] The juices studied can be arranged in the following order according to a decreasing FRAP value: blackcurrant > orange > apple. Similar order of juices was found by others.[24] The high antioxidant activity of blackcurrant juice may possibly be attributed to a high content of polyphenols present in the blackcurrant berries.

Tea polyphenols, especially the catechins, manifest antioxidant effects in humans.[25] The antioxidant activity of tea in the present study varied from 0.346 ± 0.06 mmol/100 ml in the red tea to 1.072 ± 0.19 mmol/100 ml in the white tea. This finding can be associated with the tea composition, particularly tea polyphenols, affected by the fermentation process. Green and white teas are not fermented, while during the fermentation process of fresh tea leaves, some catechins are oxidized or condensed to larger polyphenolic molecules. This fact can explain lower polyphenol content in fermented teas than in the green and white teas. Among beverages, the lowest FRAP (0.216 ± 0.05 mmol/100 ml) and polyphenol content (31 ± 1 mg/100 ml) was observed for beer, though some authors indicate that raised consumption of beer can be responsible for considerable polyphenol intake.[6]

Chocolate

Cocoa beans and chocolate are rich in polyphenols, particularly in catechins and epicatechins. Earlier studies demonstrated that the consumption of chocolate was associated with an increase in the serum antioxidant activity.[26] Antioxidant activity varies depending on a type of chocolate. In the current study, the FRAP values were decreasing in the following order: dark chocolate > semisweet chocolate > milk chocolate > white chocolate (Table 2).

Table 2  Total polyphenol content and antioxidant activity in chocolate in order of the FRAP antioxidant activity

Chocolates	N	Polyphenol content (mg/100 g)	FRAP (mmol/100 g)
Dark chocolate ^a	3	1617 ± 161	14.67 ± 1.61
Semisweet chocolate ^b	3	1483 ± 202	11.83 ± 1.89
Milk chocolate ^c	3	515 ± 79	3.80 ± 0.41
White chocolate	3	222 ± 60	0.550 ± 0.05
^aNot less than 46% total dry cocoa solids.
^bNot less than 35% total dry cocoa solids.
^cNot less than 25% total dry cocoa solids.
N = number of samples.

Among all of the chocolates tested, dark chocolate showed the highest FRAP antioxidant activity (14.67 ± 1.61 mmol/100 g) and the highest polyphenol content (1617 ± 161 mg/100 g), whereas the white chocolate presented about a 30-fold lower antioxidant activity (0.550 ± 0.05 mmol/100 g) and a 7-fold lower polyphenol content (222 mg/100 g). The antioxidant activity of chocolates seems to be related to the contents of cocoa solids. The dark chocolate tested, which contained at least 46% of dry cocoa solids, showed the highest antioxidant activity among various types of chocolates. The semisweet chocolate (containing not less than 35% of the total dry cocoa solids) had a slightly lower FRAP value than the dark chocolate. Milk chocolate, containing 25% of cocoa solids, had the lowest polyphenol content and antioxidant activity among chocolates containing cocoa solids. White chocolate, which does not contain cocoa mass, showed a very low antioxidant activity as compared to chocolates containing the cocoa solids. The polyphenol content in our study was in agreement with a study by Waterhouse et al.[27] for milk chocolate and about 50% higher in dark chocolate. These findings may be associated with the higher content of total dry cocoa solids in dark chocolates in our study.

Nuts and Seeds

The FRAP activities and polyphenol contents of analyzed nuts and seeds are shown in Table 3. In the present study, the walnuts had the highest FRAP values (55.91 ± 16.88 mmol/100 g) among the nuts and seeds group. The extremely high antioxidant capacity of walnuts is not surprising considering their high polyphenol content (3529 ± 1137 mg/100 g). Pellegrini et al.[28] reported similar results, and observed that walnuts had the highest FRAP value among the Italian nuts. Recent findings indicated that walnut consumption was associated with a reduced oxidative stress, showing protective properties against coronary heart disease.[29]

Table 3 Total polyphenol content and antioxidant activity in nuts and seeds in order of the FRAP antioxidant activity

Nuts and seeds	N	Mean dry mass content (%)	Polyphenol content in dry mass (mg/100 g)	Polyphenol content in fresh mass (mg/100 g)	FRAP in dry mass (mmol/100 g)	FRAP in fresh mass (mmol/100 g)
Walnuts	3	94.5 ± 0.6	3733 ± 1190	3529 ± 1137	59.17 ± 17.6	55.91 ± 16.88
Sunflower seeds	3	97.0 ± 0.8	1400 ± 90	1358 ± 81	12.58 ± 0.6	12.21 ± 0.48
Hazelnuts	3	93.7 ± 2.9	550 ± 130	516 ± 127	4.78 ± 1.4	4.49 ± 1.32
Pistachios	3	97.0 ± 0.8	642 ± 50	622 ± 45	1.73 ± 1.1	1.679 ± 1.03
Peanuts	3	96.2 ± 1.5	460 ± 90	442 ± 81	1.57 ± 0.1	1.507 ± 0.12
Pumpkin seeds	3	89.8 ± 6.4	140 ± 20	125 ± 14	0.95 ± 0.1	0.851 ± 0.07
N = number of samples.

The lowest FRAP activity and the polyphenol content were found in pumpkin seeds (0.851 ± 0.07 mmol/100 g and 125 ± 14 mg/100 g, respectively). Among the seeds, the sunflower seeds showed the highest FRAP activity (12.21 ± 0.48 mmol/100 g). In the northeastern regions of Poland, where the research was conducted, sunflower seeds are very popular snacks, and their consumption may possibly positively influence the antioxidant status of a diet. The antioxidant FRAP activity of foods was positively related (r = 0.99, P < 0.01) to the mean polyphenol content in all studied foods, which was in agreement with previous studies.[13] This result shows an important role of polyphenols in foods for the generation of antioxidant activity.

CONCLUSION

The results obtained in the present study showed that the studied foods were characterized by various antioxidant activities. In connection with this finding, these foods can be categorized in groups of low, medium, and high FRAP activities: <5 mmol/100 g/100 ml: tea, coffee, drinking chocolate, hot cocoa, fruit juices, wine, beer, milk and white chocolate, hazelnuts, pistachios, peanuts, pumpkin seeds; 10–15 mmol/100 g: dark and semisweet chocolate, sunflower seeds; >50 mmol/100 g: walnuts.

ACKNOWLEDGMENTS

The technical assistance of Ms. Hanna Olechnowicz is gratefully acknowledged.